Standardization of exhaled breath condensate: effects of different de-aeration protocols on pH and H2O2 concentrations

Abstract

Exhaled breath condensate (EBC) analysis is a non-invasive method to repeatedly evaluate airway inflammation. Dissolved carbon dioxide contributes to lowering EBC pH which is reversed by degassing with argon. Hypothetically, argon may also improve biomarker stability by removing reactive gases, since many markers are pH sensitive or easily oxidized. In this study, the influence of two degassing methods was assessed on (i) the volume of EBC, (ii) the EBC pH, and (iii) the concentration of H2O2 in EBC. EBC was collected from 13 healthy subjects and 12 chronic obstructive pulmonary disease subjects over 20 min, then aliquoted and either left on ice or de-aerated with argon by bubbling or surface delivery at 400 ml min−1 for 0 to 600 s, to quantify the EBC volume loss and the efficiency of pH equilibration. Biomarker stability was measured by H2O2 concentration. Both degassing methods reached a pH equilibrium by 300 s. Bubbling reached pH equilibrium faster (60 s versus 300 s), while having significantly less EBC volume loss (bubbling 3.32 ± 1.31% versus surface 10.74 ± 1.46%, p < 0.0001). The H2O2 concentration was higher in non-degassed samples (0.47 ± 0.18 µM, p = 0.017) but similar in the bubbling and surface degassed samples (0.30 ± 0.08 µM versus 0.31 ± 0.10 µM, p = 0.54). The optimal degassing methods were to bubble the aliquots with argon at 400 ml min−1 for 60 s or surface degassing for 300 s. Both methods resulted in significantly less EBC volume loss than the commonly adopted method of bubbling for 10 min. There was a significant difference in the H2O2 concentration between the degassed and the non-degassed samples.